ASMT)-overexpressed melatonin-enriched dairy goats were successfully generated through the use of pBC1-ASMT expression vector construction and prokaryotic embryo microinjection. Results: These transgenic goats have the same normal phenotype as the wild-type goats (WT). However, the melatonin levels in their blood and milk were significantly increased (p < 0.05). In addition, the quality of their milk was also improved, showing elevated protein content and a reduced somatic cell number compared to the WT goats. No significant changes were detected in the intestinal microbiota patterns between groups. When the animals were challenged by the intravenous injection of E. coli, the ASMT-overexpressed goats had a lower level of pro-inflammatory cytokines and higher anti-inflammatory cytokines compared to the WT goats. Metabolic analysis uncovered a unique arachidonic acid metabolism pattern in transgenic goats. Conclusions: The increased melatonin production due to ASMT overexpression in the transgenic goats may have contributed to their improved milk quality and enhanced the anti-inflammatory ability compared to the WT goats.Background: Transgenic animal production is an important means of livestock breeding and can be used to model pharmaceutical applications. Methods: In this study, to explore the biological activity of endogenously produced melatonin, Acetylserotonin-O-methyltransferase ( Melatonin affects immune system function and the nervous system through endocrine effects. . MelatonAANAT) and Acetylserotonin-O-methyltransferase (ASMT) are considered to be rate-limiting enzymes [AANAT-overexpressed dairy goats, high levels of endogenous melatonin render their anti-inflammatory ability by regulating autophagy processes [ASMT is the last rate-limiting enzyme in melatonin synthesis and also plays an important role in melatonin synthesis. For example, the upregulation of ASMT expression in the pineal gland of rats is associated with increased melatonin synthesis [ASMT gene demonstrates significant polymorphism, and its gene promoter and first exon polymorphism may be related to mental disorders [ASMT is involved in the improvement of reproductive performance in goats [Melatonin synthesis in organisms involves several enzymes. Among them, the arylalkylamine N-acetyltransferases  metabolism in the anti-inflammatory response of transgenic animals compared to WT animals. The results will provide novel information that will allow us to further understand the biological traits modified by melatonin-enriched transgenic technology in animals, particularly in goats.In the current study, we explored the effects of ASMT gene was cloned from the pineal gland of a goat for the construction of mammary gland-specific expression vector pBC1-ASMT (ASMT-overexpressed dairy goats and the WT goats (p > 0.05) (The Coding DNA Sequence (CDS) region of the BC1-ASMT B. SaII aBC1-ASMT C. A totaBC1-ASMT A. PCR seBC1-ASMT D,E. Ther > 0.05) .ASMT-overexpressed goats were significantly higher than that of the WT goats (p < 0.05)  (p < 0.05) (ASMT-overexpressed and WT goats (p < 0.05) (The melatonin levels in the blood and milk of the  < 0.05) A. The mi < 0.05) B,C and t < 0.05) D. There  < 0.05) E–H.ASMT-overexpressed goats, 16S sequencing was performed to detect their fecal microbiome distribution. With the increase in the effective sequence depth, the dilution curve of the sample increased rapidly and then leveled off, which indicated good sequencing quality (ASMT-overexpressed dairy goats and the WT goats (p > 0.05) (ASMT-overexpressed dairy goats and the WT goats (p > 0.05)  C. β-dive > 0.05) D. Furthe > 0.05) E. The Ky > 0.05) F. Thus, ASMT-overexpressed dairy goats in terms of their inflammatory response, 500 µL of E. coli (107/mL) was injected into the jugular vein. As a marker of inflammatory response, the C-reactive protein (CRP) increased rapidly within 1 h and then gradually decreased. The CRP level in the ASMT-overexpressed dairy goats was significantly lower than that in the WT goats (p < 0.05) (p > 0.05)  (p > 0.05) (ASMT-overexpressed goats than it was in the WT goats (p < 0.05) (To explore the  < 0.05) A. No sig > 0.05) B. The le < 0.05) C. No sig > 0.05) D,E. The  < 0.05) F.E. coli injection and then decreased gradually in both groups. The level of the proinflammatory cytokines in the ASMT-overexpressed goats was significantly lower than that in the WT goats (p < 0.05) (E. coli injection and decreased after 12 h. The levels of the anti-inflammatory factors in the ASMT-overexpressed goats were significantly higher than they were in the WT goats 1–12 h after E. coli injection (p < 0.05)  A–C. The  < 0.05) D,E.E. coli injection, the Partial Least Squares Discriminant Analysis (PLS-DA) method was used to analyze the serum metabolomic alterations. The confidence level of the PLS-DA data was above 95%, and different groups were in different ellipses. For the analysis of the positive ions, two principal component analysis groups were obtained (ASMT-overexpressed dairy goats and the WT goats (p < 0.05). Heat map analysis also showed significant differences in the metabolic composition and abundance between the groups (p < 0.05)  E.p < 0.05) . Furthermore, the up-regulated and down-regulated genes were enriched for KEGG function. After the enrichment of the up-regulated gene pathways, arachidonic acid metabolism, choline metabolism in cancer, retrograde endocannabinoid signaling, and insulin resistance showed significant differences between the groups  D in the was 1.21 E. N-acetegulated A. The diegulated B. Deoxyce groups C.AANAT transgenic goat models have been generated by using somatic cell nuclear transfer. AANAT-overexpressed goats with high endogenous melatonin levels showed good anti-inflammatory ability when challenged by LPS [ASMT is the last rate-limiting enzyme in the melatonin synthetic pathway [ASMT-overexpressed dairy goats were successfully produced. In addition, several biological traits of these transgenic goats were investigated, including the quality of their milk. In fact, these dairy products make up an important part of the human diet and are a part of a healthy life. Goat milk is more similar to human milk in terms of its nutritional factors than cow milk is [ASMT will only be expressed in the mammary gland. As predicted, the level of melatonin in milk is significantly increased in transgenic goats compared to in WT goats. This indicates that the construction of the model of ASMT mammary gland-specific-expression transgenic goats was successful. To evaluate the quality of the milk, milk composition analysis, which is a commonly accepted index that is used to evaluate the milk quality [ASMT overexpression to produce high-quality goat milk.Using transgenic technology to produce livestock with targeted traits has become an important research area in molecular breeding . Prokaryd by LPS . ASMT is pathway . In this milk is . The num milk is . The hig milk is . However milk is . Melaton milk is . pBC1-AS quality , was per quality . In addiASMT-overexpression goats has significantly increased levels of circulating melatonin. This is not surprising since lipophilic melatonin molecules can diffuse freely from the mammary gland into circulation. As a result, it can also increase the melatonin level in the gut. Due to the fact that melatonin involves the gut–microbiome–immune axis and regulates the biorhythm through intestinal bacterial activity signals mediated by the NF-Kβ pathway [ASMT overexpression would influence the gut microbiota was also investigated. It was reported that melatonin supplementation reprograms the structure of the intestinal microflora and improves the circadian rhythm of the intestinal microflora in mice fed with a high-fat diet [ASMT overexpression has little effect on the normal microbial community in the gut.In the current study, we observed that mammary-specific  pathway , whetherfat diet . HoweverE. coli was injected into the goats to induce inflammation. It has been reported that this injection induces serious inflammatory reactions and triggers brain and liver cell death in chickens [E. coli injection induced an immediate increase in CRP, an inflammation marker in animals. However, this increase in CRP was significantly suppressed in the ASMT-overexpressed goats compared to in the WT goats. The most impressive observations were that the serum proinflammatory cytokines IL-1β, TNF-α, and IL-6 were significantly reduced while the anti-inflammatory cytokines IL-10 and IFN-β were significantly increased in the ASMT transgenic goats compared to in the WT goats after E. coli challenge. These cytokine alterations may be the primary contributors to the anti-inflammatory activity of transgenic goats. On other hand, the altered fatty acid metabolism in the ASMT-overexpressed goats may also contribute to anti-inflammatory improvement. PLS-DA analysis indicates that the arachidonic acid (AA) metabolism pathway is the most significant pathway for the enrichment of upregulation factors in ASMT-overexpressed goats. AA and its derivatives are involved in immune metabolic and inflammatory reactions in organisms [Another that was focused on was the anti-inflammatory activity of melatonin-enriched transgenic goats since melatonin is also a potent anti-inflammatory molecule. For this purpose, chickens  and incrchickens . In the rganisms . It has rganisms . Prostagrganisms . PGE2, Prganisms . Melatonrganisms . TXB2 isrganisms . PhysiolASMT transgenic and WT goats, and the transgenic phenotype also does not influence the normal distribution of the gut microbiota. Our results show that the ASMT overexpression with increased endogenous melatonin will involve in the overreaction of inflammation induced by intravenously injected E. coli. This observation is consistent with previous reports that melatonin only suppresses the overaction of inflammation and can be used in serious infectious diseases [In the current study, we observed that there are no large phenotype differences between diseases . Other mASMT-overexpressed dairy goats for the first time. These goats can produce melatonin-enriched, high-protein, and low somatic cell milk compared to WT goats. The anti-inflammatory effect of the transgenic goats is also significantly improved. This is indicated by the reduced levels of proinflammatory and increased levels of anti-inflammatory cytokines after E. coli injection in transgenic goats compared to in WT goats. The underlying mechanism is related to their elevated melatonin production, which is induced by the overexpression of ASMT since melatonin is a potent anti-inflammatory molecule. The limitation of the study is the relatively small numbers of the transgenic goats due to the common practical reasons for large animal studies. Thus, this study can be considered as a concept-proof study. Based on the data from this study, we will design further studies with more samples in the near future. Another area of interest that we have noticed is whether the novel biological traits found in these transgenic goats can pass to their offspring. This is another goal for our future studies. Furthermore, using transgenic animals to improve milk quality may be an alternative way to replace antibiotic treatment. As noted earlier, this is a new area to be explored, and more and well-designed studies will be required to address unsolved problems in the future.In the study, we generated ASMT gene were amplified by PhantaTM super-Fidelity DNA polymerase . The PCR product was cloned into the pMDTM19-T vector  and was sequenced according to the manufacturer’s instructions. Primers were designed using Premier 5.0, and the same restriction XhoI site was added to the ASMT (mRNA KC290950.1) Capra hircus sequence from the National Center for Biotechnology Information (NCBI). A pBC1 Milk Expression Vector Kit  was used as the carrier skeleton. ASMT was derived from the T-vector by the XhoI restriction enzyme and was cloned into pBC1 to construct the vector pBC1-ASMT.Total RNA was extracted from the pineal gland of goat using Trizol reagent , and cDNA synthesis was performed using a cDNA synthesis kit . The complete open reading frame (ORFs) and some upper and downstream non-coding regions of the ASMT-overexpressed goats is shown in ® CIDR® Sheep and Goat Device, Auckland, New Zealand) for estrus synchronization. Excellent robust rams were used for artificial semen collection. Superovulation and endoscopic-assisted insemination were carried out on embryo-donor goats. The plasmids were digested with NotI and SalI enzymes, and the linearized DNA was extracted from the gel and was purified with a DNA purification kit . The linearized DNA solution was then injected into the cytoplasm of the prokaryotic embryos at a concentration of 10 µg/mL and a volume of 5 pL. A total of 3 to 5 embryos that were in good condition were transferred to one recipient within one hour. A total of 55 recipients were transplanted. After 60 days, a B-ultrasound was performed to examine the pregnancy status of the recipient. A total of 18 recipients became pregnant, and 10 lambed.The production process for the DNA was extracted from the ear tissue of the progeny to identify its transgenic status. Specific primers were designed using Premier 5.0 . The linASMT-overexpressed dairy goats who were 12 months of age. An amount of 5 mL of goat jugular vein blood was collected, placed at 37 °C for 30 min, centrifuged at 3000 r/min for 10 min, and the serum was stored at −20 °C. Milk sample collection followed the same procedure as “Milk quality analysis”. Blood and milk samples were mixed with methanol in a 1:4 portion and were then oscillated in a vortex. After centrifugation , the supernatant was collected and filtered into with a microporous membrane for use. Then, melatonin detection was carried out in the central laboratory of the Beijing Institute of Animal Science, Chinese Academy of Agricultural Sciences  using a high-performance liquid mass spectrometer .Blood samples were collected from Milk was collected for 3 consecutive days after 7 days of lactation. Before collection, the milk residues in the nipple were removed and cleaned. Milk with normal color, smell, and viscosity was collected at 16:00 every day. An amount of 40 mL fresh milk was mixed with 2–3 drops of saturated potassium dichromate solution and was stored at 2–7 °C. Milk composition determination was conducted based on the National Dairy Standards Accreditation Laboratory of Beijing Animal Husbandry and Veterinary Station . The somatic cell count (SCC) was determined by Fossomatic TMFC , which was based on flowcytometry. Milk protein, fat, and dry matter were measured by MilkoScan FT+ , which was based on Fourier transforinfrared spectrum analysis.ASMT-overexpressed dairy goats that were 12 months of age. Rectal feces were collected in a sterile environment and were frozen with liquid nitrogen. The samples were sequenced by 16S rDNA in Jinweizhi Biotechnology Co., Ltd . For the microbiome DNA extraction method, the PowerSoil DNA Isolation Kit  (Omega DNA Kit) was used. Primers 338F (5′-ACTCCTACGGGAGgCAGCAGcag-3′) and 806R (5′-GGACTACNNGGG TATctaat-3′) were used to amplify the V3–V4 region of the bacterial 16S rRNA gene. The clean reads of all samples were clustered and classified into the same OTUs with an identity of 97% similarity using the software . The RDPC lassifier Bayesian algorithm was used for taxonomic analysis, and the community composition of each sample was counted at a certain level. The reference database was the 16S-Silva_132 16S rRNA database . The rarefaction curve of the Alpha Diversity and PCoA Unifrac distance matrix of the Beta Diversity were calculated using Qiime (version 1.9.1). PICRUSt was used to predict the functional capacity of the microbial community.Fecal samples were collected from E. coli K12 strain DH5α were cultured in Luria–Bertani (LB) broth at 37 °C. The bacterial growth phase was measured using the absorbance of the bacterial suspension at 600 nm. An optical density (OD) of ~ 0.4 ensured that the bacteria were in the logarithmic growth phase. The number of bacteria was counted by plate counting through serial 10-fold dilutions of the inoculum to the LB agar. The bacteria were suspended and diluted to a concentration of 1 × 107 cells/mL in 0.9% normal saline for use.The E. coli (107/mL) was injected into the jugular vein of the goats. The blood was collected after E. coli injection from the jugular vein for physiological, biochemical, immune factor, and metabolomics analysis. The serum was isolated for the different analyses.The healthy goats were divided into WT and transgenic groups . An amount of 500 µL of n = 3) and from the wild type goats (n = 3) were measured. The blood was collected at 0 h, 1 h, 6 h, 12 h, 24 h, and 48 h after E. coli injection from the jugular vein. An amount of 5 mL of goat jugular vein blood was collected. An amount of 3 mL of blood was placed at 37 °C for 30 min and centrifuged at 3000 r/min for 10 min, and the serum was used directly to detect the biochemical parameters. The globulin (Glob) content was measured using an Automatic dry biochemical analyzer . And amount of 2 mL of whole blood samples was used for hematological measurements using an automatic hematology analyzer . The numbers of neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), and mononuclear macrophages were recorded. CRP was measured using a hypersensitive C-reactive protein assay kit(B2072). The sample detecting procedure was carried out in accordance with the instructions. The intra-batch coefficient of variation (CV) for the kit was 10%, and the inter-batch coefficient of variation (CV) was 15%.The physiological and biochemical parameters of the blood taken from the transgenic goats  following the manufacturer’s instructions. This method has been successfully used previously to detect the cytokines of mammals including mice  and goatThe serum sample collection at 1 h was the same as “Analysis of physiological and biochemical indexes”. Serum (100 uL) was mixed with 0.4 mL of a pre-cooled methanol solution containing 20 μg/mL 2-chloro-L-phenylalanine as the internal standard. The mixed sample was ultrasonicated for 30 min , was placed at −20 °C for 30 min, and was then centrifuged for 15 min . The supernatant was collected and dried with nitrogen. The sample was resuspended in 100 µL complex solution (acetonitrile: water = 1:1). The mixture was centrifuged at 13,000 rpm for 10 min at 4 °C, and 100 μL of supernatant was transferred to the auto-sampler vials for HPLC-MS/MS analysis. A quality control (QC) sample was prepared by mixing aliquots from all supernatant samples (10 μL from each sample). The method was validated by analyzing the pooled QC sample. Chromatographic separation of the metabolites was performed on a Thermo UHPLC system equipped with an ACQUITY UPLC HSS T3 . The sample injection volume was 2 µL, and the flow rate was set to 0.4 mL/min. The column temperature was maintained at 40 °C. The mass spectrometric data were collected using a Thermo UHPLC-Q Exactive HF-X Mass Spectrometer equipped with an electrospray ionization (ESI) source operating in either positive or negative ion mode. After the UPLC-MS analyses, the raw data were imported into the Progenesis QI 2.3  for data analysis. The preprocessing results were fed into Simca-P  for the multivariate statistical analysis.p < 0.05 was considered statistically significant.The Data were expressed in the form of mean ± standard error. One-way and Two-way variance (ANOVA) were performed followed by Duncan’s test using SPSS software, version 25.0 . The melatonin and milk quality data were analyzed by One-way ANOVA. The physiological and biochemical indicator and inflammatory factor data were analyzed by Two-way ANOVA. Additionally, time was the independent variable, and physiological and biochemical indicators and the inflammatory factors were the dependent variables. ASMT-overexpressed dairy goats. These goats can produce melatonin-enriched, high-protein, and low somatic cell milk compared to WT goats. Additionally, the anti-inflammatory effect of the transgenic goats also improved significantly. Endogenous high expression of melatonin may improve the anti-inflammatory ability of transgenic dairy goats by affecting arachidonic acid metabolism.In conclusion, we generated


No changes in HCy and vitamin D were observed. Our results demonstrate the possible role of TXB and BDNF in the pathophysiology of DD and the benefits of omega-3 FA supplementation. The study was registered with the ISRCTN registry (ISRCTN81655012).In the DEPOXIN project, we have found that a high ratio of omega-6/omega-3 fatty acids (FA) is associated with worsening of depressive symptoms in children and adolescents with depressive disorder (DD) and that the 12-week omega-3 FA supplementation modulates DD symptoms. Here we present our results of the secondary outcomes: the levels of thromboxane (TXB), brain-derived neurotrophic factor (BDNF), homocysteine (HCy) and vitamin D. Fifty-eight patients were randomized into two arms. One group received a fish oil emulsion enriched with omega-3 FA, and the other received a sunflower oil emulsion containing omega-6 FA, for 12 weeks. Depressive symptoms were evaluated, using the Child’s Depressive Inventory (CDI). The patients with DD had elevated TXB levels and decreased vitamin D levels, as compared to healthy controls. Both CDI and omega-6/omega-3 ratio correlated positively with TXB and negatively with BDNF at baseline. Compared to the omega-6 FA group, the supplementation with omega-3 FA for 12 weeks significantly reduced plasma TXB ( Depressive disorder (DD) is a serious global issue in children and adolescents, with a prevalence of about 5.7% among those aged 13–18 years old .Core symptoms for childhood depression include persistent sadness, loss of interest and enjoyment, and increased fatigability . The patOver the years, the diet ratio of omega-6 fatty acids (FA) to omega-3 FA has changed from 1:1 to 15–20:1. ,8,9. SucPolyunsaturated fatty acids are involved in the regulation of inflammation and fluidity of membranes and may contribute to the pathophysiology of DD. Arachidonic acid , which is formed by elongation/desaturation of linoleic acid , is a substrate for the synthesis of eicosanoids, such as prostacyclins, thromboxanes, and prostaglandins , stimulating proinflammatory cytokine synthesis. The eicosanoids are formed by the catalytic action of cyclooxygenase (COX). On the other hand, omega-3 fatty acids, such as eicosapentaenoic acid  and docosahexaenoic acid , are metabolized by COX and lipoxygenase to polyhydroxy metabolites, e.g., D-resolvins formed from DHA, or to trihydroxy metabolites, e.g., E-resolvins formed from EPA, both with anti-inflammatory properties. For these reasons, the ratio between omega-6 and omega-3 FA plays an important role in the regulation of inflammatory homeostasis . An incrp = 0.017), and after three months of omega-3 FA supplementation, this ratio dropped to 7.6/1 in DD patients.In our recent work , we demoSeveral studies have examined plasma/urine levels of thromboxane B (TXB), to determine the pro-inflammatory platelet activation as an indirect marker of inflammation in the body ,18. It wBrain-derived neurotrophic factor (BDNF) is one of the growth factors, neurotrophins. Deficiency in neurotrophins in adults may contribute to hippocampal pathology during the development of depression. BDNF regulates a key transcription factor that is involved in neuroprotection, neuroinflammation, and cellular growth . HoweverEvidence suggests that homocysteine (HCy) level is associated with depression, vascular diseases, and disorder of neurotransmission in adults. In adults, an association between the higher concentration of HCy and increased risk of depression was found. It was calculated that the reduction of HCy by 0.19 mg/L could decrease depression by about 20% . Little Vitamin D is known to be important for the calcium and phosphorous metabolism and bone health, but it is also assumed to have an effect on mental health . VitaminIn our DEPOXIN project , we have demonstrated the effect of fish oil emulsion rich in omega-3 FA on depressive symptoms in children and adolescents with DD (the primary outcomes) ,40. The Our results should contribute to the understanding of the interrelationships between the observed parameters in children and adolescents with depressive disorder.Subjects, inclusion and exclusion criteria, study design and intervention, randomization, patient enrollment, and treatment have been described in detail in our previous works ,40.Briefly, we enrolled thirty-one outpatients with depressive disorder (DD) and twenty-nine outpatients with mixed anxiety and depressive disorder (MADD) in our study  .Twenty healthy children and adolescents were enrolled to the healthy control group.Patients were randomized into two arms (ratio 1:1) and received daily 20 mL of either fish oil emulsion rich in omega-3 FA (Om3 group) or a comparator of sunflower oil emulsion rich in omega-6 FA (Om6 group) for 12 weeks, alongside their standard antidepressant therapy . The fish oil emulsion comprised 2400 mg of total omega-3 FA , and the sunflower oil emulsion contained 2467 mg of omega-6 linoleic acid. Both emulsions were prepared by Cultech Ltd., Port Talbot, UK.Patients were advised to follow their standard diet and report any changes in their dietary habits throughout the study. The self-rated scale of Children’s Depression Inventory (CDI) was used to rate the depression severity ,42. Moreg, 10 min) within 1 h of collection, and the obtained serum and EDTA-plasma were stored at −80 °C, until further analysis.Patients and healthy controls provided the venous blood samples after a 12 h overnight fast. The blood samples were centrifuged , according to the manufacturer’s protocol. The concentration of thromboxane is presented in pg/mL.BDNF was determined in plasma with a Human BDNF ELISA kit , according to the manufacturer’s protocol, and is expressed in ng/mL.Homocysteine was determined in plasma by Advia Centaur XP HCZ kit  in the Advia Centaur system . The homocysteine level is expressed in µmol/L.Serum vitamin D was determined by a 25-OH Vitamin D total Elisa kit . The vitamin D concentration is expressed in ng/mL.t-test or Wilcoxon signed-rank test were used to compare data between weeks of intervention, depending on the normality of the data. A non-parametric Mann–Whitney U test was used for the evaluation of difference between patients and healthy controls. The effect of omega-3 fatty acids on the studied parameters following 12 weeks of supplementation was evaluated with the two-way ANOVA test. The Spearman rank correlation coefficient was used for the correlation determination.Data are presented as the mean ± standard deviation (SD) for the normally distributed variables or as a median and interquartile range for data showing departures from normality. Student’s p < 0.05 was considered statistically significant. Data analyses were performed, using StatsDirect® 3.3.4  and IBM SPSS Statistics 25.0.0. The StatsDirect® program was used for graphical representation of the data.A value of The results of the effect of omega-3 FA on the depressive disorder symptom severity (the study primary outcome) were published by Trebatická et al. . Brieflyp < 0.001 for both), compared to the healthy controls . Following the omega-3 FA supplementation, EPA and DHA levels increased to 462% and 270% of baseline [Basal serum levels of EPA (1.08 ± 0.61 µg/0.1 mL) and DHA (2.53 ± 1.02 µg/0.1 mL) were significantly lower  in either depressed males  or depressed females . However, a positive correlation was found between BMI and the serum omega-6/omega-3 FA ratio in depressed patients , but not in the control group .We analyzed the correlation among depressive symptoms rated as a CDI score  and BMIp < 0.001 and p = 0.031, respectively). We evaluated possible differences in basal levels of BDNF, CDI, omega-6/omega-3, EPA, and DHA between patients already treated and initially diagnosed, and no significant difference was found for any parameter. There was no significant difference in the BDNF and HCy levels between all patients and healthy controls.The plasma TXB, BDNF, HCy, and serum vitamin D levels at the baseline are shown in p = 0.027 and HCY: 10.4 ± 2.7 μmol/L, p < 0.0001). The statistically significant gender differences in BDNF and HCy levels were not observed in the healthy controls.However, we found strong gender differences in BDNF and HCy levels at the baseline . DepressThe effect of omega-3 FA supplementation on the studied parameters is shown in p = 0.194), and at week 12 of supplementation, to 70.9% of baseline (p = 0.0374). Compared to baseline (100%), TXB values reached 100.3% and 98.7% , following omega-6 FA administration after 6 and 12 weeks (p = 0.009) and after 12 weeks to 129.9% of baseline (p = 0.04)  .p = 0.024) and BDNF (p = 0.011) levels were observed between Om3 and Om6 groups after 12 weeks of supplementation (Significant differences in TXB (entation . No signp = 0.021). The TXB showed only a borderline significant treatment effect (p = 0.091).A significant treatment effect of omega-3 FA, compared to omega-6 FA, with two-way ANOVA, in BDNF, was confirmed after 12 weeks of intervention (The correlations between severity of depression (CDI score) or omega-6/omega-3 ratio and investigated parameters for patients with DD or healthy controls at baseline are presented in We evaluated the correlations of DHA and EPA levels with TXB and BDNF in all patients (FA levels were published in detail in our prior work ) Figure. Surprisn = 30) (DD without MADD) and mixed anxiety and depressive disorder , only EPA but not DHA correlated positively with BDNF in patients in the DD subgroup. Patients with MADD did not show a correlation between these parameters .The primary outcome of the DEPOXIN project was to investigate the effect of omega-3 FA on the severity of depression as determined by the CDI score, compared to the effect of omega-6 FA. We were the first to present a relationship between the omega-6/omega-3 FA ratio and the depression severity in children and adolescents .We found in DD patients a positive correlation between BMI and omega-6/omega-3 ratio, but not between BMI and CDI. This contradicts the findings in a Swedish study where the authors found that obesity is associated with a risk of anxiety and depression independent of other factors (neuropsychiatric disorders and low socioeconomic status of the family) . Howeverp < 0.001) independent of gender.The secondary outcome of this trial was the comparison of the levels of thromboxane, brain-derived neurotrophic factor, homocysteine, and vitamin D in the pediatric population with DD and healthy controls, as well as the investigation of the effect of the omega-3 FA supplementation alongside the standard antidepressant therapy on depressive symptoms. A significant increase of TXB levels by 299% was observed in the group of depressed children and adolescents, compared to healthy controls (Thromboxane B2 (TXB2) is a marker of platelet activation. It is formed from AA under catalytic activity of cyclooxygenase (COX) and could be considered as an indirect marker of activated inflammatory pathways .p = 0.03) in the same patient cohort. These findings support the conclusions of DiNicolantonio and OKeefe [Consistent with the conclusions of Colasanto et al. , TXB levd OKeefe , that and OKeefe ,40 sugged OKeefe  resultinp ≤ 0.001) in our pediatric patients. In contrast to the findings of Pallavi et al. [p = 0.014) and females (p = 0.009). We have also detected a negative correlation of BDNF with omega-6/omega-3 ratio (p = 0.038) in all patients. We realize that assessing the situation in the brain through systemic markers may not always be accurate. Similarly, the pathway of BDNF into the blood cannot be inferred from a human study, unless its two-way brain–blood transport is taken into account. BDNF synthesis is thought to be transcriptionally promoted by DHA [Brain-derived neurotrophic factor (BDNF) is a protein that is implicated in the pathophysiology of depression. We did not find altered plasma BDNF levels in our pediatric patients, compared to healthy controls, contrary to the results of Pandey et al. , who foui et al. , who cond by DHA . Howeverd by DHA . On the  p = 0.00. We have p = 0.00. We havep < 0.001), but not in the healthy controls. Chung et al. [Homocysteine (HCy) is a non-proteinogenic sulfurated amino acid . No diffg et al.  explaineg et al. . Howeverg et al. . HCy is g et al. . HoweverThe 12-week supplementation with the omega-3 FA or omega-6 FA did not have an impact on HCy levels. As our results do not lead to a clear conclusion regarding the association between HCy and the depression severity in children and adolescents, or the influence of gender, more studies with a well-defined diagnosis of DD are needed to confirm the findings.p = 0.031), but our healthy control group also had an insufficient level of vitamin D (<30 ng/mL). Libuda et al. [Some observational studies highlighted an inverse association between serum vitamin D levels and depressive symptoms in adults . On the a et al.  did not a et al. . Howevera et al.  found thWe did not determine any of the direct markers of inflammation  in depressed children and adolescents, for technical reasons. Patients had hsCRP levels in the physiological range. We used an indirect marker, thromboxane, to indirectly monitor the inflammatory response.The food diaries were not collected during the study. The patients were advised to maintain their normal diet and avoid the consumption of any fatty acid supplements or antioxidants throughout the study.We are aware of a small number of males , as compOur findings suggest the potential involvement of thromboxane and brain-derived neurotrophic factor (BDNF) in the pathophysiology of depressive disorder in children and adolescents. A significant positive correlation was found between the depression severity or omega-6/omega-3 FA ratio and plasma thromboxane B and a negative correlation with BDNF. The children and adolescents with depressive disorders had higher levels of thromboxane B and reduced levels of vitamin D, when compared to healthy controls. The supplementation with omega-3 FA alongside the standard antidepressant therapy may have a beneficial effect on thromboxane levels. However, the positive effect of omega-3 FA supplementation on BDNF levels was seen only in depressed patients diagnosed with depressive disorder, but not in patients with mixed anxiety and depressive disorder . These findings are encouraging and need to be confirmed in a larger study of the pediatric population.


Moreover, high IL-1α  and low IL-27  greatly increased the risk of ending up in the severe group. This model was especially sensitive in order to predict critical status . Furthermore, high levels of HGF and IL-1α showed significant results in the survival analysis . HGF, IL-1α, and IL 27 at hospital admission were strongly associated with severe/critical COVID-19 patients and therefore are excellent predictors of bad prognosis. HGF and IL-1α were also mortality biomarkers.Pneumonia is the leading cause of hospital admission and mortality in coronavirus disease 2019 (COVID-19). We aimed to identify the cytokines responsible for lung damage and mortality. We prospectively recruited 108 COVID-19 patients between March and April 2020 and divided them into four groups according to the severity of respiratory symptoms. Twenty-eight healthy volunteers were used for normalization of the results. Multiple cytokines showed statistically significant differences between mild and critical patients. High HGF levels were associated with the critical group (OR = 3.51;  In December 2019, a new strain of coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was recognized to have emerged in Wuhan, China. Along with SARS-CoV . After a+ T cells, including effector, memory, and regulatory T cells 3 .To impute cytokine values below the assay detection limit, robust regression on order statistics was used: this method performs a regression to impute low values assuming log-normal quantiles for samples with a detection rate of at least 20%, after checking that the data follow a log-normal distribution. To accomplish this, the non-detects and data analysis (NADA) R package was used  . MoleculThe strength of each biomarkers was evaluated at the individual level to determine the pulmonary severity of the patient. The main variable was severity, which is an ordinal variable with four levels. The first model to be fitted was an ordinal logistic regression model or proportional odds model . To confirm this, the proportional odds model was compared with a multinomial logistic regression one through the likelihood ratio test. However, in none of the cases was it possible to assume this hypothesis, so multinomial models were fitted.Biomarkers associated with the severity at the 10% significance level were identified as potential biomarkers and they were evaluated simultaneously to fit a multivariable model.The leave-one-out-cross-validation (LOOCV) procedure was used to estimate the prediction accuracy of the final fitted models, and receiver operation characteristic (ROC) curve analysis was used to assess their discriminate ability. The final models were evaluated according to the area under the ROC curve (AUC). In addition, sensitivity and specificity were obtained by setting an optimal threshold.p < 0.05. The cut-off point is established in each cytokine selecting the one with the greatest area under the ROC curve (AUC) in the individual model.A survival analysis was also performed with the final panel of cytokines identified by the multivariable models. The outcome was tested related to T2 . For survivals, the days of follow-up were hospitalization time or 28 days in outpatients after leaving the hospital. The Kaplan-Meier survival function was used by the log-rank test to determine differences in survival rates, considered different when n = 34, 31.5%, IC 95% (23.07–41.23)], (ii) moderate , (iii) severe , and (iv) critical  defined by their need of oxygen supplementation.Our cohort had a median age of 67 years, mostly male (63.26%). The control group of healthy volunteers had a median age of 61 years and most of them (57.1%) were also male. Patients were divided into four severity degrees based on the subsequent outcome during their hospital stay: (i) mild . In a similar manner, if IL-1α  or IL-27  were over- or under-expressed at admission, the risk of being in the severe group was 1.36 and 1.74 respectively (1/0.5753) referred to as the mild group.The best multivariable model based on these molecules is the one with four cytokines: HGF, IL1a, IL2, and IL27 . The sex7) imply a 25% chance of being critical while levels above 223 pg/mL increase that critical risk up to 50%. On the contrary, patients with HGF levels below 64 pg/mL (26) have no risk  of ending up critical. In the same manner, low IL-1α levels at admission had a probability over 37% of being mild, while IL-1alpha levels over 1024 pg/mL (210) had 50% chances of being in the severe group. Last, but not least, lower levels of IL-27 at admission were also associated with the severe group since level under 1 are reflected in a 50% chance of belonging to the severe group while IL-27 levels over 64 pg/mL (26) decrease that risk to practically 0%.The fitted models are used to estimate the predicted probabilities and their associated confidence bands of severity group. These estimated probabilities are visualized as effect plots in Internal validation by the LOOCV procedure shows that AUC is significantly greater than 0.5 in all severity groups , especiap = 0.033 and p = 0.011 respectively).Here we have described, after performing a 45-plex cytokine array on plasma samples from 108 patients at hospital admission, that five cytokines are statistically significantly different according to the degrees of severity in COVID-19. Indeed, high levels of HGF and IL-1α coupled with low levels of IL-27 at admission can predict bad clinical outcome referred to the patient subset with better prognosis, being especially important the high level of HGF as predictors of admission in intensive care units. Moreover, this multivariate model was especially sensitive in order to identify those patients who end up in a critical status  following hospital admission. Last, but not the least, we have also described how the combination of high levels of HGF IL-1 α at admission can predict mortality, showing significant results in the survival analysis , IL-1 (Anakinra), and TNFα  . The REMWe are aware of the main limitations of our study. (i) Our study did not include a large sample size. Perhaps, we should have performed previously a statistical power analysis. Nevertheless, our sample size is consistent with previous reports ,24,25,30Our study characterized the plasma cytokine profile of COVID-19 patients at hospital admission, based on their subsequent clinical evolution into four well-defined degrees of severity, revealing that HGF, IL-1α, and IL27 were strongly associated with disease severity and could be used as excellent predictors of bad prognosis. Indeed, HGF and IL-1α are also mortality biomarkers. Therefore, the early detection of HGF, IL-1α, and IL27 plasma levels in patients in COVID-19 patients can provide useful information for getting quickly intensive treatment as well as providing possible therapeutic targets.


We have systematically investigated the effects of hydrogen annealing on Ni- and Al-contacted carbon nanotube field-effect transistors (CNTFETs), whose work functions have not been affected by hydrogen annealing. Measured results show that the electronic properties of single-walled carbon nanotubes are modified by hydrogen adsorption. The Ni-contacted CNTFETs, which initially showed metallic behavior, changed their p-FET behavior with a high on-current over 10 µA after hydrogen annealing. The on-current of the as-made p-FETs is much improved after hydrogen annealing. The Al-contacted CNTFETs, which initially showed metallic behavior, showed unipolar p-FET behavior after hydrogen annealing. We analyzed the energy band diagrams of the CNTFETs to explain experimental results, finding that the electron affinity and the bandgap of single-walled carbon nanotubes changed after hydrogen annealing. These results are consistent with previously reported ab initio calculations. ΦM = 5.1 eV and has the technological advantage of good wetting between the Pd and the carbon nanotube [2 gate dielectrics, where Sc has a low work function of ΦM = 3.3 eV [Carbon nanotube field-effect transistors (CNTFETs) are being extensively studied for various applications, including microprocessors, high-frequency electronics, and display electronics ,2,3,4. Ananotube . Zhang e= 3.3 eV .Reduction in a single-walled carbon nanotube’s work function due to hydrogenation has been reported theoretically ,10 and e3 hybridization and opened the bandgap. However, these results are a more significant effect than the experimental results of CNTFETs [2 layer were converted to n-FETs after hydrogen annealing [2O3-capped CNTFETs after thermal annealing in a vacuum due to doping [Only a few experimental studies have reported the effects of hydrogen on CNTFETs; however, these results are contradictory ,18,19. Ao doping . The effI–V) characteristics of Ni- and Al-contacted CNTFETs. Ni has a high work function of 5.2 eV, similar to that for Pd, and Al has a lower work function of 4.3 eV. Previous studies reported [This paper presents experimental results on the effect of hydrogen annealing on the current–voltage . The SiO2 layer was thermally grown and the HfO2 layer was deposited by atomic layer deposition (ALD). The carbon nanotubes used in this work were grown by thermal chemical vapor deposition (CVD) using a combination of Fe and Ge nanoparticles as previously described [2 layer. The 30 nm-thick SiO2 layer was deposited on HfO2 by plasma-enhanced CVD (PECVD) and densified at 950 °C. The top PECVD-SiO2 layer was then implanted with 5 × 1015 cm−2, 20 keV Ge, and annealed in nitrogen at 600 °C for 40 min to create Ge nanoparticles [2 layer was then removed using an HF vapor etch to expose the Ge nanoparticles on the HfO2 layer. Then the substrate was dipped in ferric nitrate solution for 1 min and rinsed with hexane. The CNT growth was carried out using CVD in a hot-wall reactor at atmospheric pressure. CNTs were grown at 850 °C for 20 min using a mixture of methane (1000 sccm) and hydrogen (300 sccm) immediately after a pre-anneal in hydrogen (1000 sccm) at 900 °C.A p+ Si substrate (0.005 Ω∙cm) was employed as a back gate. After a standard cleaning process, we formed a gate dielectric stack of SiOescribed . The nanarticles . The topBack-gate both Ni and Al-contacted CNTFETs were fabricated using the lift-off technique. The metals were deposited by sputtering, and the source/drain electrodes were formed using direct-write optical lithography and lift-off. The gap between the source/drain electrodes was 2 µm and the width was 5 µm, as shown in I–V measurements were carried out before and after hydrogen annealing using an Agilent 4155C semiconductor parameter analyzer . We used a Cascade REL3200 probe station  for the room temperature measurements and a Nagase BCT-43MDC probe station  for the low-temperature measurements. The room temperature measurements were carried out in the air. The low-temperature measurements were carried out in a vacuum of 1.3 × 10−4 Pa after the samples were kept in a high vacuum for 30 h to exclude the effects of water and oxygen.The synthesized carbon nanotubes were characterized using field emission scanning electron microscopy , high-resolution transmission electron microscopy , and Raman spectroscopy . TEM sample preparation consisted of scraping the sample’s surface with a surgical blade and transferring it onto a carbon-coated Cu grid. Raman spectra were obtained using a micro-Raman system with He-Ne laser excitation (632.8 nm) at a power of 12 mW. 2/HfO2 gate dielectric and Ni source/drain contacts. The area density of carbon nanotubes is low, and almost all carbon nanotubes are isolated.RBM (cm−1) = 248/d (nm) [E = 0.9/d (nm) [8/d (nm) . The dia9/d (nm) . The banI–V measurements on the CNTFETs gave more than 80 functional devices each for Ni- and Al-contacted devices. I–V characteristics are categorized into two main types: low-drain current with high ION/IOFF ratio and high-drain current with low ION/IOFF ratio. Hydrogen annealing has had a significant effect on the characteristics of the CNTFETs. To clarify the transformation behavior, we compare the I-V characteristics before and after hydrogen annealing.I–V behavior has gate voltage modulation of the drain current (gating effect) after hydrogen annealing, giving rise to the improved on-current. The top-gated graphene FETs have similar sub-threshold characteristics because of a zero or very narrow bandgap [ bandgap .I–V characteristics with various current conductions that are associated with the bandgap variation of carbon nanotubes. The Al-contacted devices showed p-FET behavior after hydrogen annealing. Hydrogen annealing improved the on-current and leakage current. The device with higher current conduction turned into a p-FET with a lower ION/IOFF ratio. The large leakage current is attributed to the tunneling current across the narrow bandgap. The device with lower current conduction turned into p-FET behavior with the steep sub-threshold slope with a higher ION/IOFF ratio. The on-current of the Al-contacted CNTFETs is smaller than that of the Ni-contacted CNTFETs. As for Schottky barrier transistors, on-current is determined by the Schottky barrier height. Higher work function metals, such as Ni and Pd, form a lower Schottky barrier height; consequently, they induce higher on-current. Thus, these results suggest that the fabricated devices are relevant to Schottky barrier transistors.VG) dependence of the gate leakage current density (JG) of the Al-contacted CNTFETs after hydrogen annealing. As the gate leakage current at 3 V is about three orders of magnitude smaller than the drain current of the CNTFET with a higher ION/IOFF ratio, the drain current is coming from the carbon nanotubes.SS = (dlogID/dVG)−1. The sub-threshold slope is temperature independent and constant around 140 mV/dec. When the tunneling current through the Schottky barrier is dominant in drain current, the sub-threshold slope has temperature dependence. This is because the tunneling current has temperature dependence. The sub-threshold slope of temperature-independent and lower values, less than 200 mV/dec, indicate that the lower Schottky barrier height is formed at the contact region [The steep values of the sub-threshold slope of the Al-contacted CNTFETs suggest that Al/CNT contact has a lower Schottky barrier after hydrogen annealing. We measured the temperature dependence of the sub-threshold characteristics in a high vacuum to test this possibility. t region ,32. As ft region .−4 Pa for 30 h (ION) and ION/IOFF ratio before and after hydrogen annealing for the Ni-contacted CNTFETs. In all cases, hydrogen annealing leads to a significant increase in on-current, especially in the devices with an ION/IOFF ratio less than 100, which initially showed metallic behavior. The excellent correlation between on-current and ION/IOFF ratio indicates that the bandgap of carbon nanotubes determines on-current. The increase in on-current after hydrogen annealing could reduce the contact resistance and the reduced electron affinity of carbon nanotubes. Higher on-current over 10 µA is a result of the creation of a bandgap in carbon nanotubes. Narrow bandgap FETs exhibit high on-current due to increased mobility, and a small ION/IOFF ratio due to the large tunneling leakage current.Earlier research on Al-contacted CNTFETs has shown either p-type or n-type FET behavior ,35,36. Tfor 30 h . The CNTEg and χ are bandgap and electron affinity of carbon nanotubes, and ΦM is the work function of Ni. The estimated electron affinity and work function of the pristine carbon nanotubes were 4.2 and 4.5 eV, respectively [ION/IOFF ratio and a higher on-current. As for the CNTFETs with semiconducting carbon nanotubes, the increase in bandgap improves on-current.To explain the effects of hydrogen annealing on the CNTFETs, the energy band diagrams of the Ni- and Al-contacted CNTFETs before and after hydrogen annealing are considered. It should be mentioned that the work functions of Ni and Al remain unaffected by hydrogen annealing, which differs from Pd. ectively . These vectively . The banAs a final remark, we have also examined the effect of hydrogen annealing on CNTFETs with Pd source/drain contacts. However, we could not obtain any results because all the Pd films came off after hydrogen annealing; this was a result of hydrogen-induced plastic deformation.ION/IOFF ratio, due to creating a narrow bandgap in carbon nanotubes. The Al-contacted CNTFETs, which initially exhibited metallic behavior, were converted into unipolar p-FET behavior. We have introduced new energy band diagrams for the CNTFETs after hydrogen annealing. It is necessary to reduce electron affinity and widen the bandgap of carbon nanotubes to explain experimental results. These variations of parameters are supported by the ab initio calculations.In this paper, the effects of hydrogen annealing on CNTFETs have been systematically investigated using Ni- and Al-contacted CNTFETs. It emerges that the electronic characteristics of the CNTFETs are modified by hydrogen annealing, which can change the band structure of carbon nanotubes due to the presence of covalent C–H bonds. The Ni-contacted CNTFETs, which initially exhibited metallic behavior, were converted into p-FET behavior with a higher on-current and lower


To reduce incision complications, minimally invasive operative approaches for treatment with acute Achilles tendon rupture have been developed, such as Mini-open repair and percutaneous repair. Which technique is the better surgical option? In the present study, we compared the two surgical procedures— modified Mini-open repair versus percutaneous repair—in the treatment of acute Achilles tendon rupture.From January 2016 to November 2018, 68 matched patients with acute Achilles tendon rupture were divided into treatment group (Mini-open with modified Ma-Griffith technique) and control group (the Ma–Griffith technique). The patients were then treated with different surgical techniques and followed up for no less than 24 months, and the functional outcome scores and complications were retrospectively evaluated.P=0.147). The Mini-open repair group showed reliably higher American Orthopedic Foot and Ankle Society (AOFAS) Ankle-Hindfoot Score and Achilles tendon Total Rupture Score (ATRS) than the control group in functional assessment . There was no cases of sural nerve injury in Mini-open repair group, whereas the percutaneous repair group had 5 cases of the same (P=0.027). No significant differences were found in the calf circumference (32.3±3.9 vs. 31.8±3.6) (P=0.564), range of motion of the ankle , or wound complications (34/0 vs. 34/0) (P=1.000) between the two groups at the end of the follow-up time. However, the percutaneous repair group had a shorter average operating time (23.1±5.2 min) than that of the Mini-open repair group (27.7±4.3 min) (P=0.000).The mean follow-up time in Mini-open repair group was 29.0±2.9 months, and that in control group was 27.9±2.9 months (Acute Achilles tendon ruptures may be treated successfully with a new Mini-open repair system or percutaneous repair technique. However, the Mini-open repair system may represent a superior surgical option, since it offers advantages in terms of direct visual control of the repair, AOFAS Ankle-Hindfoot Score, Achilles tendon Total Rupture Score and risk of sural nerve palsy.Case-control studies, Level of evidence, 3.The online version contains supplementary material available at 10.1186/s12891-021-04802-8. Acute rupture of the Achilles tendon is one of the most common types of tendon ruptures in the human body . This tyTo reduce incision complications, minimally invasive operative approaches have been developed, such as percutaneous repair and Mini-open repair. Percutaneous suture technique is widely used by many surgeons in Achilles tendon repair, but sural nerve injury remains a problem. Sural nerve entrapment is one of the most common complications after percutaneous surgery –9. The cWhich technique is the better surgical option for treatment with acute Achilles tendon rupture? In the present study, we used a new Mini-Open Achilles tendon repair system with modified Ma-Griffith technique Fig.  22, 23], 2322, 2This control-matched study was conducted at the Navy General Hospital of PLA. Using the database and the medical records (between January 2016 and November 2018), 34 patients with acute Achilles tendon rupture treated with Mini-open repair were matched to 34 patients treated with percutaneous minimally invasive anastomosis. The age(±5), sex, and BMI(±5) were similar and well-matched in both groups. This study approved by the ethical committee of the Navy General Hospital of PLA in 2016. All experiments were performed in accordance with relevant guidelines and regulations. Both the surgical interventions described in study were implemented as standard-of-care at hospital. The patients can withdraw from the study at any time without discrimination or retaliation, and the medical treatment and rights and interests will not be affected. Regardless of any patient who refuses to participate in the study, he/she will still receive appropriate surgical treatment. Sample-size estimation was based on what was needed to detect the difference in complications in the groups. We estimated that more than 30 patients in each group were enough to detect a 20% difference in AOFAS score or ATRS between groups, with the alpha set at 0.05 and beta at 0.1. An additional 10 % of total participants was planned for each group to make up for possible loss. All patients read the detailed information sheet and signed a written consent form.The criteria for inclusion in this study were as follows: (1) Patients with acute, closed Achilles tendon rupture; (2) A positive Thompson test; (3) Presence of pitting as assessed by observation and palpation between the two broken ends of the Achilles tendon; and (4) Complete rupture of the Achilles tendon as observed by ultrasonic examination. By contrast, patients with incomplete rupture of the Achilles tendon or open injury, patients with a repair time of more than 2 weeks, and patients with incomplete clinical data were excluded.Percutaneous Achilles tendon repair was performed in this study in accordance with the Ma–Griffith technique Fig. . The patEstablishment of the surgical incision. The patient was placed in the prone position, and epidural anesthesia was applied. The end of the Achilles tendon was subsequently exposed by making an approximately 2–3 cm transverse incision at the level of tendon rupture  Non–weight-bearing equinus cast in place (0–2 weeks after surgery). A cast in a 20° to 25 ° “equinus” position was applied after both procedures and a below the knee gravity equinus cast was applied for approximately 2 weeks.“Walker boot” period/muscle strength recovery period (3–10 weeks after surgery). After 2 weeks, the below the knee cast was removed and the patient began to ambulate in a “walker boot”, range-of-motion movements for the ankle were practiced, the leg muscles were strengthened, and normal gait was gradually restored. During the next 3-4 weeks the angle of the “walker boot” was gradually changed to a neutral position. The “walker boot” period was maintained for at least 8 weeks.Muscle strengthening period (10 weeks after surgery and beyond). Ten weeks after surgery, ankle flexibility and leg muscle strength were improved to increase the stability of the lower limbs and to gradually restore motor function. Attempts were made to achieve full movement at the ankle. The ATRS , 28 and The patients were followed up for no less than 24 months, and the functional outcome scores and complications were retrospectively evaluated. Functional evaluation was based on the clinical AOFAS score and ATRS along with other findings, such as the length of the scar, neurologic deficit, calf circumference, and range of motion of the ankle. Whether there was no deep vein thrombosis or sural nerve injury was based on color Doppler ultrasound and electromyography.P value of less than 0.05 was considered significant.SPSS 23.0 was used for statistical analysis. The two groups were compared with respect to sex, age, follow-up time, operating time, hospital stay, calf circumference, AOFAS score, ATRS score, number of wound complications, sural nerve injury, and ankle ROM. Statistical analysis was conducted by an independent statistician not directly involved in the study. The Paired Samples t-Test, the results of which were expressed as the mean and standard deviation (SD), was used for the quantitative data analysis with equal variance assumed between the two groups. The Chi-square test was used to assess the qualitative data between the two groups. A The baseline information and demographics of both groups are listed in Table P=0.147). The Mini-open repair group showed reliably higher AOFAS Ankle-Hindfoot Score and ATRS than the control group in functional assessment . There was no cases of sural nerve injury in Mini-open repair group, whereas the percutaneous repair group had 5 cases of the same (P=0.027). No significant differences were found in the calf circumference (32.3±3.9 vs. 31.8±3.6) (P=0.564), range of motion of the ankle , or the number of wound necrosis or infection (34/0 vs. 34/0) (P=1.000) between the two groups at the end of the follow-up time. However, the percutaneous repair group had a shorter average operating time (23.1±5.2 min), compared with the Mini-open repair group (27.7±4.3 min) (P=0.000). No cases of sural nerve injury in the Mini-open repair group were reported, but five such cases were found in the percutaneous repair group (P=0.027).The follow-up data were summarized, and functional results were evaluated in both groups Table . All patMultivariate analysis was performed to analyze the relationship. The age, BMI, Operating time, hospital stay, ATRS and ROM of ankle joint were taken as independent variables, while AOFAS was taken as dependent variables for linear regression analysis. The results were shown in the Supplement Tables 1 and 2.p= 0.001), suturing technique (p= 0.002) [The choice of treatment for acute Achilles tendon rupture remains a challenge for surgeons. Despite hundreds of publications in the medical literature on the subject of acute rupture of the Achilles tendon, its optimal treatment remains under debate. One study from the Netherlands has described that although open repair 65%) was the most common surgical technique and Bunnell sutures (55%) were mostly applied, trauma surgeons and orthopaedic surgeons differed significantly on surgical technique (= 0.002) . Surgica= 0.002) . However= 0.002) . Minimal= 0.002) , 35. Ma = 0.002) . Mini-op= 0.002) . Khan, R5% was th= 0.002) . An incr= 0.002) , 39, Ach= 0.002) , or the = 0.002) , 40, 41 in vitro model via a progressive rehabilitation program. Mini-open repair can achieve reliable suture strength with fewer stitches and knots, as strong as that of the open Krackow restoration, but weaker than those of the Achillon and PARS techniques. To a certain extent, the greater tensile strength of the suture used, the stronger the tensile strength of the Mini-open repair suture structure. Therefore, the repaired Achilles tendon exhibits high tensile strength, allows early functional exercise, and requires skills that can be easily mastered with a short learning curve. This Mini-open repair system has reduced suture knots, lowering the foreign body sensation of the suture knot and keloid after recovery and consequently improving the function and appearance of hindfoot. Our results suggest that both Mini-open repair and percutaneous repair can achieve satisfactory functional outcomes in patients with Achilles tendon. However, the Mini-open repair group showed reliably higher AOFAS Ankle-Hindfoot Score and ATRS than the control group in Function assessment . The functional results are comparable to the results in several other investigations using open and mini-open repair techniques [2 value of in the model is 0.699 and it meant that the age, BMI, Operating time, hospital stay, ATRS and ROM of ankle joint could explain 69.9 % variations in AOFAS. The model was tested by the F-test , indicating that at least one of age, BMI, operating time, hospital stay, ankle range of motion and ATRS has an impact on the AOFAS Score. In the percutaneous repair group, the R2 value of model was 0.619 and it also passed the F-test , indicating that the age, BMI, a model could explain 61.9% of the variation in AOFAS.In the present study, we used a new minimally invasive and direct visual control of Achilles tendon suture system Fig. . This Michniques –47. Caldchniques . Chen etchniques , and 90.Sural nerve palsy is one of the most important complications of minimally invasive repair of Achilles tendon rupture. A recent meta-analysis  suggestsThis study has certain limitations in control-matched designs. Selection bias was not avoided, considering that the surgical treatment to be performed was determined by the orthopedic surgeon. Although the two groups of patients were matched, only the sex, age and BMI of the patients were matched in the groups to maintain a sufficient number of patients.In conclusion, our results suggest that satisfactory functional outcomes can be obtained for both treatment methods. This new Mini-open repair system is easy to operate and the guide instrument of the system is placed deep into the paratenon, preventing the risk of a subcutaneous nerve being trapped in the suture itself. Mini-open repair may be the superior surgical option, given its advantages in terms of direct visual control of the repair, AOFAS Ankle-Hindfoot Score, ATRS and risk of sural nerve palsy.Additional file 1: Supplement Table 1. Multivariate analysis results in Mini-open repair group


Improved diagnostics by positron emission tomography/computed tomography (PET/CT) can lead to adjustments in treatment procedures . We analyzed the impact of 68Ga-PSMA-11-PET/CT. All patients had a PSA ≤0.5 ng/ml. An experienced radiation oncologist determined the radiotherapy concept, first with consideration of the PET/CT, second hypothetically based on the clinical and pathological features excluding PET/CT results.We retrospectively analyzed 76 patients with biochemical recurrence after radical prostatectomy in whom SRT was planned after 68Ga-PSMA-11-PET/CT was found in 54% of the patients. The median pre-PET/CT PSA level was 0.245 ng/ml (range 0.07–0.5 ng/ml). The results of the PET/CT led to a change in the radiotherapeutic target volume in 21 patients (28%). There were major changes in the target volume including the additional irradiation of lymph nodes or the additional or exclusive irradiation of bone metastases in 13 patients (17%). Minor changes including the additional irradiation of original seminal vesicle (base) position resulted in eight patients (11%).Without considering the PET/CT, all 76 patients would have been assigned to RT, 60 (79%) to the bed of the prostate and seminal vesicles alone, and 16 (21%) also to the pelvic lymph nodes because of histopathologic risk factors. Uptake indicative for tumor recurrence in 68Ga-PSMA-11-PET/CT for radiation planning, a change in the treatment concept was indicated in 28% of patients. With PET/CT, the actual extent of the tumor can be precisely determined even with PSA values of ≤0.5 ng/ml. Thus, the treatment concept can be improved and individualized. This may have a positive impact on progression free survival. Our results warrant further prospective studies.Using  Radical prostatectomy (RP) is considered to be a standard treatment option for patients with clinically localized prostate cancer (PCa). Nevertheless, up to 50–80% of these men develop biochemical recurrence depending on risk factors such as an advanced pathological stage, a high Gleason score or positive surgical margins . In caseAt PSA levels <1 ng/ml, most imaging methods are not suitable to detect the correlate for disease progression. Therefore, up to 20% of patients with SRT to the prostate bed  without morphological correlate will be treated locally without actual local recurrence (68Ga-PSMA has been used since 2012 as PSMA-ligand in recurrent prostate cancer (68Ga-PSMA-11-PET/CT is significantly higher in comparison to other imaging methods.Prostate-specific membrane antigen (PSMA) is a cell surface protein with high expression in majority of prostate cancer . 68Ga-PSe cancer –9. EspecIn a retrospective analysis of 2,533 patients with biochemical progression after RP, Afshar-Oromieh et al. found that 69% of the patients had at least one positive lesion indicating PCa recurrence. The detection rates were 43% for PSA levels ≤0.2 ng/ml, 58% for PSA >0.2 to ≤0.5 and 72% for PSA >0.5 to ≤1.0. Tumor detection was clearly associated with PSA level and higher Gleason scores . Overall, 76 patients were found to fulfill the inclusion criteria. For all patients SRT was planned after An experienced radiation oncologist determined patient’s actual treatment concept with consideration of the PET/CT images. Retrospectively a hypothetical treatment concept (prescription and planning target volume [PTV] contouring) was planned based on the clinical and pathological parameters excluding PET/CT results: In pT2- and pT3a-tumors the PTV included the prostate bed and the basis of the former seminal vesicles. In pT3b-tumors, the bed of seminal vesicles was included, too. Inclusion of regional pelvic lymph nodes was considered in case of histopathologic risk factors .All patients gave their written informed consent for a retrospective analysis of their data in an anonymized form. The study was approved by the local ethics committee of Ulm University (221/20-FSt/Sta).68Ga-HBED-CC-PSMA complex  was produced as already published. For labeling the 50 mCi , iThemba LABS, South Africa 68Ge/68Ga radionuclide generator was used . Intravenous contrast  and 15 to 20 mg of furosemide were administered in 71 (93%) and 68 patients (89%) unless contraindicated. For a diagnostic CT, scans were performed 70 s past contrast injection for the venous phase.PET/CT images were acquired by a Biograph mCT (40)S in 3D acquisition mode 63.4 ± 11.4 min after intravenous infusion of 160.3 ± 29.4 MBq A tracer uptake more than the immediate surrounding tissue and not related or explained due to the physiological expression was considered as pathologic. Two experienced nuclear medicine physicians with more than 10 years of experience in PET/CT analyzed the images.The median PSA level before PET imaging was 0.245 ng/ml (range 0.07–0.5 ng/ml). Median age was 67 years (range 47–79 years). Patient characteristics are shown in 68Ga-PSMA-11-PET/CT was found in 54% of the patients.Pathological tracer uptake as a sign of tumor recurrence in 68Ga-PSMA-11-PET/CT lead to adaptation of RT planning in 28% (n = 21) of cases. In the hypothetical scenario without considering PET/CT results all the76 patients would have received RT: 60 (79%) to the bed of the prostate and seminal vesicles alone, and 16 (21%) also to the pelvic lymph nodes because of histopathologic risk factors.Additional information from We have defined major and minor changes. Major changes included the additional or exclusive irradiation of lymph nodes or the additional or exclusive irradiation of bone metastases based on the PET/CT. Minor changes included the additional irradiation of original seminal vesicle (base) position Figure 1Based on the PET/CT, in six patients (8%), bone metastases were detected and were additionally irradiated with median 45 Gy (range 39–54 Gy).PSA values 6 months after completion of SRT were available in 54 patients, out of which 47 (87%) patients achieved a PSA response Figure 468Ga-PSMA-11-PET/CT led to changes of the RT target volume in 28% of patients with PSA ≤0.5 ng/ml after RP.In our retrospective study Detecting the site of prostate cancer recurrence is crucial for a successful treatment planning. In the presence of distant metastases, a prostate bed RT is not indicated. On the other hand, in the event of an exclusive loco-regional recurrence, a long-term ADT could be avoided or at least delayed by SRT. In the pre-PSMA era, most patients with PSA-failure had to undergo “blind” SRT under the assumption of local recurrence limited to the prostatic bed only.It is equally imperative to stress that a negative PSMA-PET should not delay early SRT as the sensitivity of PSMA PET/CT for detection of micrometastases is questionable. Therefore, in the absence of any suspicious findings on PSMA-PET/CT, early SRT of the prostate bed should be offered without time delay .68Ga-PSMA-11-PET/CT led to changes of the radiation concept in 28% of all cases. All patients had a PSA <0.5ng/ml, impressively demonstrating the great potential of this imaging approach.On the other side of the spectrum, the possibility to visualize PSMA avid lesions at an early stage prior to the SRT offers the unique possibility of individualization of treatment planning as is shown in our study. The impact of PSMA-PET/CT on SRT planning is extensively evaluated in several studies, albeit in heterogeneous patient population as is evident from 68Ga-PSMA-11-PET/CT was positive in 41 patients (34.4%). Pathological PSMA uptake was detected in the prostate bed (three patients), in the pelvic lymph nodes (21 patients), in the retroperitoneal lymph nodes (four patients) and in bone (21 patients). The initial planned radiation concept was changed in 36 patients (30.2%) due to the PET/CT results.Our results are in line with the retrospective study published by Farolfi et al. , to our In contrast, a multicenter post-hoc analysis of 270 patients with biochemical recurrence after RP with a PSA ≤1 ng/ml (median 0.48 ng/ml) showed that PSMA-PET/CT had a major impact in 19% of patients . A major68Ga-PSMA-11-PET and its impact on RT procedure in 100 patients after radical prostatectomy. Median PSA level was 1.0 ng/ml (range 0.12–14.7 ng/ml). 29 patients had initial pN1 disease. In 76 patients, at least one pathological PSMA uptake was found. Of these, 80% showed no morphological correlate in the corresponding CT or MRI. The tumor stage was changed in 43% of the patients. Due to the PSMA-PET/CT imaging, initial RT planning was modified in 59% of all cases. An additional simultaneous integrated boost to the prostate bed or lymph nodes was given to 32 and 63%, respectively. Ten patients received stereotactic body radiation therapy to single bone metastases  or PET scan prior to SRT planning. The primary endpoint is the biochemical progression-free survival after SRT .In the future, the superior soft-tissue contrast of PET/MRI may be able to further improve the detection of pelvic tumor lesions. First results showed that the detection rate was higher than the published results for PET/CT . Up to n68Ga-PSMA-11-PET/CT showed a high impact on radiation therapy procedure in patients with biochemically recurrent prostate cancer at PSA levels <0.5 ng/ml. With 28% changes in radiotherapy planning, 68Ga-PSMA-11-PET/CT is an important tool in guiding radiation treatment in this patient group. However, clinical data about the outcome of those treated patients have to be awaited.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All patients gave their written informed consent for a retrospective analysis of their data in an anonymized form. The study was approved by the local ethics committee of Ulm University (221/20- FSt/Sta).RT, TW, and AB contributed to conception and design of the study. JM, RT, and DBo organized the database. DBo performed the statistical analysis and wrote the first draft of the manuscript. JM, TK, DBa, MB, CB, AB, VP, and TW wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.